PROJECT SUMMARY While many organizations, including but not limited to big pharma, use nanotechnology to formulate the delivery of therapeutic nucleic acids (TNA), the number of concepts approved for clinical use is only a handful. The major reason for this is the general lack of understanding of TNA properties critical for their immunocompatibility. Recently, there have been press releases announcing several US biotech companies dropping TNAs due to severe inflammatory reactions (cytokine storm) in patients. To address the issue, some of the companies switched to more sophisticated formulations that employ rationally designed nucleic acids (nano-TNAs). It is evident that the immunotoxicity and immunomodulatory effects of new nano-TNAs are largely unknown and must be defined to permit successful translation of this technology into the clinic. This project will inform the scientific community about immunogenicity of nano-TNAs and provide a guide for tuning their physicochemical properties to avoid undesirable immunological side effects. The current application proposes to investigate the immunogenicity of nano-TNAs and to propose a strategy for their successful transition to therapeutic applications. Based on the data from our previous work and preliminary results, we hypothesized that the immunogenicity of nano-TNAs can be controlled by changing their relative size, charge, shape and composition. To test this innovative hypothesis, we seek R01 mechanism support. In this project, Drs. Afonin?s (UNC Charlotte) and Khisamutdinov?s (Ball State University) laboratories will generate a panel of nano-TNAs and extensively characterize them. Drs. Marriott?s (UNC Charlotte) and Dobrovolskaia?s (NCL) groups will assist in studies and analysis of the immunological responses triggered by nano-TNAs with the goal of determining the structure-activity relationship (SAR) in terms of immuno- and hemato-compatibility. Dr. Lee?s laboratory, at Clemson University, will assist with further detailed in vivo studies of nano-TNAs. Dr. Tropsha (UNC Chapel Hill) will assist in development of predictive computational models based on the obtained experimental data and correlation of biological data to physicochemical properties of nano-TNAs. The results of this cutting-edge, interdisciplinary work will improve the understanding of SAR for nano-TNAs and will lead to the development of nano-TNA platforms for broader biomedical applications. We will make our results publically available via database server that will feature detailed profiles of all known nano-TNAs. Ultimately, completion of this proposal will lead us to development of efficient next generation nano-TNA platforms lacking immunogenicity and featuring high therapeutic potential. The long term goal of this study is to elevate nano- TNAs to the level of clinical use.